This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Recent evidence indicates that Notch1 signaling, a key regulator of stem cell differentiation, may play a role in preventing valvular calcification. However, the underlying mechanisms remain obscure. In addition, the roles that other members of Notch receptor family may play in this process have not been investigated, although they share the same ligands and downstream pathways with Notch1. Our aim is to investigate whether other Notch receptors and ligands may play a role in regulating osteogenesis and contribute to cardiovascular calcification. The second aim of this project is to identify a potential connection between inflammatory processes known to induce atherosclerotic lesions and calcification and the regulation of Notch signaling through post-translational modifications. Lastly, a third aim is to demonstrate the selectivity of Notch family members in activating specific downstream pathways leading to vascular calcification. In a preliminary study we investigated the potential role of Notch receptors in regulating the osteogenic differentiation of mesenchymal stem cells (MSC), as a model of vascular calcification. The expression patterns suggested that Notch2 (N2) and Notch3 (N3) might be involved in the osteogenic differentiation process. The roles of N2 and N3 were further investigated by transfecting the active intracellular domains of N2 or N3, or an empty vector (V) into mouse MSC. Osteogenic differentiation was measured after 2, 3 and 4 days via alkaline phosphatase (ALP) activity and hydroxyapatite (HA) crystal formation detected by von Kossa staining. N3-transfected MSC displayed enhanced ALP activity and HA formation compared to control MSC in either osteogenic or control media at all time-points, consistent with a role in promoting osteogenesis, as suggested by N3 expression pattern. N2-transfected MSC also showed enhanced ALP activity and HA crystal formation at 2 days. Further results, suggested an inhibitory role for N2 at later stages of osteogenic differentiation. Notch 2 and 3 also showed selectivity in activating standard downstream Notch targets such as HES transcription factors. Preliminary investigation of the mechanism responsible for N2 and N3 selectivity suggests that N2 and N3 may compete for Jag1 ligand binding. It has been previously demonstrated that Notch ligand binding is regulated by glycosylation, which inhibits Jag ligand binding and promotes Delta ligand binding. In agreement with this mechanism, our results suggest that a decrease in N3 glycosylation and an increase in N2 glycosylation could account for the shift we see in Jag1 binding, which gradually increases in N3 immunocomplexes and decreases in N2 immunocomplexes. Preliminary results, indicate that during osteogenesis there is a decrease in N3 glycosylation and an increase in N2 glycosylation. Notch glycosylation can in turn be regulated by redox mechanisms, which modify disulfide bridges and change the structure of the EGF repeats in the Notch extracellular domain. Our main objective is to confirm the preliminary results by additional identification of N2 and N3 immunoreactive protein bands and their post-translational modifications (glycosylation, nitrosylation) using mass-spectrometry. The sites of post-translational modifications will be also identified if possible, to provide additional information on the mechanism of Notch signaling. In addition, no information exists on the processing of Jag ligands during Notch signaling. Therefore another objective is to analyze Jag1 by mass spectrometry to identify potential modifications by glycosylation and proteolytic processing. We are using 1D gel electrophoresis and in-gel digestion to identify the proteins and search for post-translational modifications.